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. 2013 Jul 16;110(29):12120-5.
doi: 10.1073/pnas.1302170110. Epub 2013 Jul 1.

LNK Genes Integrate Light and Clock Signaling Networks at the Core of the Arabidopsis Oscillator

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Free PMC article

LNK Genes Integrate Light and Clock Signaling Networks at the Core of the Arabidopsis Oscillator

Matias L Rugnone et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Light signaling pathways and the circadian clock interact to help organisms synchronize physiological and developmental processes with periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Here we describe a family of night light-inducible and clock-regulated genes (LNK) that play a key role linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana. A genomewide transcriptome analysis revealed that most light-induced genes respond more strongly to light during the subjective day, which is consistent with the diurnal nature of most physiological processes in plants. However, a handful of genes, including the homologous genes LNK1 and LNK2, are more strongly induced by light in the middle of the night, when the clock is most responsive to this signal. Further analysis revealed that the morning phased LNK1 and LNK2 genes control circadian rhythms, photomorphogenic responses, and photoperiodic dependent flowering, most likely by regulating a subset of clock and flowering time genes in the afternoon. LNK1 and LNK2 themselves are directly repressed by members of the TIMING OF CAB1 EXPRESSION/PSEUDO RESPONSE REGULATOR family of core-clock genes in the afternoon and early night. Thus, LNK1 and LNK2 integrate early light signals with temporal information provided by core oscillator components to control the expression of afternoon genes, allowing plants to keep track of seasonal changes in day length.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genomewide analysis of light and clock interactions in the control of gene expression and identification of LNK genes. (A) Experimental design. Plants were grown under 12-h light/12-h dark cycles for 14 d and then exposed or not to a 1-h light pulse in the middle of the night or subjective day on the 15th day. (B) Comparative genomewide expression analysis of the effect of a light pulse given during subjective day time (x axis) vs. night time (y axis). (C) Overlap between 87 genes that are rhythmically expressed under multiple conditions (23) and 65 genes that showed a stronger induction by light during night time compared with subjective day time (Dataset S1). (D) Microarray data corresponding to the relative response of LNK genes to a 1-h light treatment given in the middle of the night or subjective day. (E) Relative expression levels of LNK1 and LNK2 measured by qRT-PCR. The analysis was conducted in WT, phyA;phyB, and cry;cry2 plants grown under 12-h light/12-h dark cycles and exposed or not to a 1-h light pulse in the middle of the night (n = 3). nd, not detectable. Data represent average + SEM. (F) Circadian expression of LNK1 and LNK2 genes. Expression was determined by qRT-PCR during the third day under free running conditions. n = 4. Data are average + SEM. White, dark, gray, and hatched boxes indicate day, night, subjective night, and subjective day, respectively.
Fig. 2.
Fig. 2.
Physiological characterization of LNK1 and LNK2. (A) Hypocotyl length of WT, lnk1, lnk2, and lnk1;lnk2 mutant seedlings grown under continuous white light (LL) (n = 6 replicates of 10 seedlings each). (B) LNK1 and LNK2 control the floral transition in plants grown under LD (16-h light/8-h dark) conditions. (C and D) Flowering time measured as the number of rosette leaves at bolting in LD (C) and SD (D) conditions (8-h light/16-h dark). ANOVA followed by a Tukey´s multiple comparison test was used to evaluate the statistical significance of differences observed between genotypes. Error bars indicate +SEM (***P < 0.001, *P < 0.05). (E and F) Circadian rhythms of leaf movement in continuous light (n = 7). Plants were grown under LD cycles and then transferred to constant light and temperature conditions. Error bars indicate +SEM. Open and hatched boxes indicate subjective day and subjective night, respectively.
Fig. 3.
Fig. 3.
LNK1, a nuclear protein, positively regulates expression of circadian genes with an afternoon phase. (A) LNK:YFP detection by confocal microscopy in the hypocotyl of seedlings expressing 35S:LNK:YFP in a WT background is shown on the left (first panel). Fluorescence following staining with DAPI is shown in blue (second panel). The merged image (white) and the image with the transmitted light channel are also shown (third and fourth panels, respectively). (Scale bar, 20 µm.) (B) Phase enrichment of circadian-regulated genes whose expression was down- or up-regulated in lnk1;lnk2 mutant compared with WT plants, according to RNA-seq data of plants grown under continuous light conditions. The phase overrepresentation analysis was conducted with Phaser (http://phaser.mocklerlab.org/) and was based on the phases of gene expression estimated from data obtained using WT plants grown under LD conditions (23). Dashed line corresponds to P = 0.01. (C) Average normalized expression of 36 genes from the cluster with the largest number of genes whose expression was altered in lnk1:lnk2 mutants compared with WT plants grown under LD conditions. Normalized expression of PRR5 (D), ELF4 (E), and FKF1 (F), three genes present in the cluster shown in C. (C–F) Plants grown under LD cycles were sampled every 4 h, starting 2 h after lights on. n = 3, Error bars indicate + SEM.
Fig. 4.
Fig. 4.
LNK1 and LNK2 are necessary for the proper function of the circadian clock. CCA1 (A), LHY (B), PRR9 (C), PRR7 (D), PRR5 (E), and TOC1 (F) mRNA expression measured by qRT-PCR in plants grown under 12-h light/12-h dark cycles and then transferred to continuous light. Values are expressed relative to PP2A and normalized to the maximum value of each gene. Data represent average +SEM (n = 4). Open and hatched boxes indicate subjective day and subjective night periods, respectively.
Fig. 5.
Fig. 5.
LNK1 and LNK2 are repressed by the TOC1/PRR1 family of circadian clock components. (A) TOC1 binds to LNK1–4 gene promoters. ChIP-qPCR assays were conducted using TOC1. Minigene (TMG) seedlings grown under 12-h light/12-h dark cycles. Samples were collected at ZT 6 and ZT16 in the light and dark, respectively. (B–E) LNK1 (B and D) and LNK2 (C and E) expression measured by qRT-PCR in continuous light relative to PP2A (n = 4). Plants were grown under 12-h light/12-h dark cycles and then transferred to continuous light. Error bars indicate +SEM. Open and hatched boxes indicate subjective day and subjective night, respectively. (F) Model showing the proposed function of LNK1 and LNK2 in the circadian clock. Light regulates LNK1 and LNK2 expression in the morning, which then act to promote, directly or indirectly (dashed line), the expression of a subset of afternoon genes, including the core clock genes PRR5 and ELF4. During the afternoon and early evening, PRR9, PRR7, PRR5, and TOC1 bind to the LNK promoters blocking their expression.

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